Expression of Phospholipase D during Castor Bean Leaf Senescence

Membrane deterioration in plant senescence is commonly associated with progressive decreases in membrane phospholipid content. This study investigated the expression and regulation of phospholipase D (PLD; EC 3.1.4.4) during senescence in castor bean (Ricinus communis L. cv Hale) leaf discs. The rate of leaf senescence was accelerated by 50 [mu]M abscisic acid and was attenuated by 50 [mu]M cytokinin during incubation at 23[deg]C for up to 5 d. Leaf senescence was indicated by decreases in the content of total proteins, chlorophyll, and phospholipids. PLD activity in both membrane-associated and cytosolic fractions showed a gradual increase in the absence of phytohormones. Abscisic acid stimulated an increase in membrane-associated PLD and had little effect on the soluble form. On the other hand, cytokinin retarded the increase in membrane-associated PLD. Immunoblotting analysis using PLD-specific antibodies revealed that the changes in PLD activity were correlated with those of PLD protein. Analysis of PLD by nondenaturing PAGE showed the appearance of a PLD structural variant, PLD 3, in abscisic acid-treated leaf discs. Northern blotting analysis using a PLD cDNA probe revealed an increase in PLD mRNA in senescing leaf discs. These data indicate complex mechanisms for the regulation of PLD during senescence, which include increases in membrane-associated PLD, differential expression of PLD isoforms, and changes in amounts of PLD protein and mRNA. Such controlled expression points to a role for PLD in membrane deterioration and plant senescence.     

Multiple Forms of Phospholipase D following Germination and during Leaf Development of Castor Bean

Multiple molecular forms of phospholipase D (PLD; EC 3.1.4.4) were identified and partially characterized in endosperm of germinated seeds and leaves of castor bean (Ricinus communis L. var Hale). The different PLD forms were resolved by nondenaturing polyacrylamide gel electrophoresis, isoelectric focusing, and size-exclusion chromatography. PLD was detected with both a PLD activity assay and immunoblots with PLD-specific antibodies. There were three major forms of PLD, designated types 1, 2, and 3, based on their mobility during nondenaturing polyacrylamide gel electrophoresis. Molecular masses of the PLD variants were estimated at 330, 230, and 270 kD for the types 1, 2, and 3, respectively. Isoelectric points of the native type 1, 2, and 3 PLDs were approximately 6.2, 4.9, and 4.8. Under the in vitro assay conditions used, the three forms of PLD exhibited the same substrate specificity, hydrolyzing phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylglycerol (PG) but not phosphatidylserine (PS) and phosphatidylinositol (PI). The three forms of PLD differed in their substrate preferences, and the order of activities was: PLD 1, PE > PG = PC; PLD 2, PE > PG > PC; PLD 3, PE = PG = PC. The Km values of PLDs 1, 2, and 3 for PC were 1.92, 2.62, and 5.18 mM, respectively. These PLDs were expressed differentially following seed germination and during leaf development. Type 1 was found in the early stages of seedling growth and in young leaves, type 2 was present in all the tissues and growth stages examined, and type 3 was expressed in senescent tissues. The PLDs shifted from largely cytosolic to predominantly membrane-associated forms during leaf development. The present studies demonstrate the structural heterogeneity of plant PLD and growth stage-specific expression of different molecular forms. The possible role for the occurrence of multiple molecular forms of PLD in cellular metabolism is discussed.     

Intracellular Localization of Jasmonate-Induced Proteins in Barley Leaves

The plant growth substance jasmonic acid and its methyl ester (JA-Me) induce a set of proteins (jasmonate-induced proteins, JIPs) when applied to leaf segments of barley (Hordeum vulgare L. cv. Salome). Most of these JIPs could be localized within different cell compartments by using a combination of biochemical and histochemical methods. Isolation and purification of various cell organelles of barley mesophyll cells, the separation of their proteins by one-dimensional polyacrylamide gel electrophoresis and the identification of the major abundant JIPs by Western blot analysis, as well as the immuno-gold labelling of JIPs in ultrathin sections were performed to localize JIPs intracellularly. JIP-23 was found to be in vacuoles, peroxisomes, and in the granular parts of the nucleus as well as within the cytoplasm; JIP-37 was detected in vacuoles and in the nucleoplasm; JIP-66 is a cytosolic protein. Some less abundant JIPs were also localized within different cell compartments: JIP-100 was found within the stromal fraction of chloroplasts; JIP-70 is present in the peroxisome and the nucleus; JIP-50 and JIP-6 accumulate in vacuoles. The location of JIP-66 and JIP-6 confirms their possible physiological role deduced from molecular analysis of their cDNA.     

ATP Citrate Lyase from Germinating Castor Bean Endosperm: Localization and some Properties

ATP citrate lyase (EC 4.1.3.8) has been found in crude extracts from endosperm tissue of germinating castor bean and shows its maximum activity in 4- to 5-day-old seedlings. A strict requirement for coenzyme A and adenosine 5′-triphosphate was demonstrated. The pH optimum for the reaction is around 7.5. The unstable enzyme can be stabilized by freezing and addition of citrate and glycerol. (−)-Hydroxycitrate is a potent inhibitor. The molecular weight is about 400,000. The adenosine 5′-triphosphate citrate lyase is localized in the plastids, where it possibly plays a role in providing acetyl coenzyme A for lipid biosynthesis.     

Sugar Uptake and Translocation in the Castor Bean Seedling II. Sugar Transformations During Uptake

Changes in the dry weights of various parts of the castor bean seedling showed that the rates of transfer of material through the cotyledons to the embryonic axis exceeded 2 mg/hour after 5 to 6 days of germination. The sugar present in the endosperm was predominantly, and in the cotyledon almost exclusively, sucrose. Anatomical features were described which contribute to the efficiency of the cotyledons as organs of absorption and transmittal of sucrose to the embryonic axis, where hexoses are much more prevalent.     

Crown Gall on Castor Bean Leaves: II. FORMATION OF SECONDARY TUMOURS

Secondary tumours were formed on the cotyledonary leaf petiole,the hypocotyl, and first true leaf of castor bean seedlingsafter inoculating the blades of the cotyledonary leaves withAgrobacterium tumefaciens. Depending on the strain of bacteriaemployed, 0 to 80 per cent of the plants developed secondarytumours. The ability of different strains to initiate secondarytumours was not obviously correlated with their relative effectivenessin initiating primary tumours. Though all produced primary tumours,five out of ten auxotrophic strains failed to yield secondarytumours, whereas only one out of 14 prototrophic strains failedto do so. Both the number of plants developing secondary tumoursand the frequency with which these tumours occurred on differentparts of the plant were positively correlated with the concentrationof the primary inoculum. Tumours also developed on the cotyledonaryleaf petiole and on the hypocotyl after vacuum infiltrationof A. tumefaciens into the blade of cotyledonary leaves. Inmost instances (9 out of 11 plants) no tumours were formed onthe blade of the infiltrated leaf. Thus, tumour formation equivalentto secondary tumours can occur in the absence of a primary tumouror an overt primary wound. Excision of inoculated leaves showedthat the stimulus for secondary tumour formation moves fromthe blade to the hypocotyl within 24 h. Attempts to demonstratethe presence of a sub-cellular tumour-initiating agent in homogenatesof inoculated leaves were unsuccessful. A. tumefaciens, however,was found in the petiole of the cotyledonary leaf and in thehypocotyl within 24 h of inoculation. The migrating agent responsiblefor secondary tumour formation in castor beans is concludedto be intact bacteria.     

Subcellular Localization of Zinc and Calcium in Bean (Phaseolus vulgaris L.) Tissues

Two bean (Phaseolus vulgaris L.) cultivars differing in growth responses to zinc were examined for differences in uptake and subcellular localization of ⁶⁵Zn during a 15-day growth period. The zinc-sensitive cultivar Sanilac showed initially a much higher rate of absorption, which declined after 24 hours. The zinc-tolerant cultivar Saginaw showed a slow but steady rate of absorption for 10 days. In roots as well as in stem callus tissues of both cultivars, three-fourths of the absorbed ⁶⁵Zn was localized in the “cytoplasmic” supernatant fractions (containing ribosomes and vacuolar sap). Very little (less than 7%) ⁶⁵Zn was localized in the cell wall fraction. There was a much greater proportion of the absorbed ⁶⁵Zn localized in root mitochondria and nuclei of the zinc-sensitive Sanilac than in the zinc-tolerant Saginaw. Stem callus tissues, however, did not show such cultivar differences in zinc accumulation at the sub-cellular level.     

An Improved Precipitation Technique for Intracellular CI- Localization in Plant Tissues3

The standard fixation medium for intracellular CI^- localizationdeveloped for animal specimens does not provide for good ultrastructuralpreservation when applied to plant tissues. This problem isovercome to a large extent by incorporation of picric acid inthe CI^- precipitation-fixation medium. The new medium is alsoapplicable to high-salt plant tissues.     

Subunit Structure of Castor Bean Hemagglutinin

Two constituent polypeptide chains of castor bean hemagglutinin (CBH-A) were isolated from the performic acid-oxidized or reduced-carboxymethylated CBH-A by chromatography on DEAE-cellulose or Sepharose 4B. From the analyses of the N-terminal amino acids, the amino acid compositions and the tryptic peptides of each chain, it was found that the larger chain with mol. wt. 34,000 and the smaller chain with mol. wt. 31,000 were homologous with the Ala and He chains of ricin D, respectively, and the subunit structure of CBH-A is represented as (α′/β′)₂ in relation to αβ of ricin D.     

Intracellular Localization of Peptide Hydrolases in Wheat (Triticum aestivum L.) Leaves

Protoplasts from 8- to 9-day-old wheat (Triticum aestivum L.) leaves were used to isolate organelles which were examined for their contents of peptide hydrolase enzymes and, in the case of vacuoles, other acid hydrolases. High yields of intact chloroplasts were obtained using both equilibrium density gradient centrifugation and velocity sedimentation centrifugation on sucrose-sorbitol gradients. Aminopeptidase activity was found to be distributed, in approximately equal proportions, between the chloroplasts and cytoplasm. Leucyltyrosine dipeptidase was mainly found in the cytoplasm, although about 27% was associated with the chloroplasts. Vacuoles shown to be free from Cellulysin contamination contained all of the protoplast carboxypeptidase and hemoglobin-degrading activities. The acid hydrolases, phosphodiesterase, acid phosphatase, α-mannosidase, and β-N-acetylglucosamidase were found in the vacuole to varying degrees, but no β-glucosidase was localized in the vacuole.     

Biosynthesis of the Macrocyclic Diterpene Casbene in Castor Bean (Ricinus communis L.) Seedlings : Changes in Enzyme Levels Induced by Fungal Infection and Intracellular Localization of the Pathway

Casbene is a macrocyclic diterpene hydrocarbon that is produced in young castor bean (Ricinus communis L.) seedlings after they are exposed to Rhizopus stolonifer or other fungi. The activities of enzymes that participate in casbene biosynthesis were measured in cell-free extracts of 67-hour castor bean seedlings (a) that had been exposed to R. stolonifer spores 18 hours prior to the preparation of extracts, and (b) that were maintained under aseptic conditions throughout. Activity for the conversion of mevalonate to isopentenyl pyrophosphate does not change significantly after infection. On the other hand, the activities of farnesyl pyrophosphate synthetase (geranyl transferase), geranylgeranyl pyrophosphate synthetase (farnesyl transferase), and casbene synthetase are all substantially greater in infected tissues in comparison with control seedlings maintained under sterile conditions. The subcellular localization of these enzymes of casbene biosynthesis was investigated in preparations of microsomes, mitochondria, glyoxysomes, and proplastids that were resolved by centrifugation in linear and step sucrose density gradients of homogenates of castor bean endosperm tissue from both infected and sterile castor bean seedlings. Isopentenyl pyrophosphate isomerase and geranyl transferase activities are associated with proplastids from both infected and sterile seedlings. Significant levels of farnesyl transferase and casbene synthetase are found only in association with the proplastids of infected tissues and not in the proplastids of sterile tissues. From these results, it appears that at least the last two steps of casbene biosynthesis, geranylgeranyl pyrophosphate synthetase and casbene synthetase, are induced during the process of infection, and that the enzymes responsible for the conversion of isopentenyl pyrophosphate to casbene are localized in proplastids.     

Biosynthesis, Intracellular Transport and In Vitro Processing of 11S Globulin Precursor Proteins of Developing Castor Bean Endosperm

In vivo labeling experiments to study the biosynthesis of 11Sglobulin in developing castor bean (Ricinus communis) endospermdemonstrated that the subunit polypeptides of the 11S globulinwere synthesized as high molecular weight precursors with heterogeneousmolecular weights. These proglobulin species were not synthesizedconcomitantly during seed maturation. The largest proglobulinwas synthesized from 20 days after anthesis, whereas the smallerproglobulins were synthesized from 30 days after anthesis. Subcellularfractionation of the pulse-labeled endosperm showed that the[³⁵S]methionine label was present in proglobulins in both theendoplasmic reticulum (ER) and dense vesicles shortly afterthe pulse labeling. The label in the proglobulin in ER decreasedduring the chase and appeared in mature globulins associatedwith crystalloids of vacuoles (protein bodies). Proglobulinsin the ER fraction prepared from the pulse-labeled developingendosperm were processed in vitro into globulins by the matrixfraction of protein bodies isolated from the dry castor bean.Overall results indicate that precursor proglobulin moleculessynthesized on rough ER are transported to vacuoles via densevesicles, and are cleaved there by the matrix protease to yieldmature globulin. ¹Department of Botany, University of Maryland, Present address:CollegePark, MD 20742, U.S.A. ²Department of Biology, Faculty of Science, Kobe University,Present address:Rokkoudai, Nada, Kobe 657, Japan (Received June 1, 1987; Accepted December 16, 1987)     

Lipase Activities in Castor Bean Endosperm during Germination

Two lipases were found in extracts from castor bean (Ricinus communis L.) endosperm. One, with optimal activity at pH 5.0 (acid lipase), was present in dry seeds and displayed high activity during the first 2 days of germination. The second, with an alkaline pH optimum (alkaline lipase), was particularly active during days 3 to 5. When total homogenates of endosperm were fractionated into fat layer, supernatant, and particulate fractions, the acid lipase was recovered in the fat layer, and the alkaline lipase was located primarily in the particulate fraction. Sucrose density gradient centrifugation showed that the alkaline lipase was located mainly in glyoxysomes, with some 30% of the activity in the endoplasmic reticulum. When glyoxysomes were broken by osmotic shock and exposed to KCl, which solubilizes most of the enzymes, the alkaline lipase remained particulate and was recovered with the glyoxysomal “ghosts” at equilibrium density 1.21 g/cm³ on the sucrose gradient. Association of the lipase with the gly-oxysomal membrane was supported by the responses to detergents and to butanol. The alkaline lipase hydrolyzed only monosubstituted glycerols. The roles of the two lipases in lipid utilization during germination of castor bean are discussed.     

Arsenic Speciation in Phloem and Xylem Exudates of Castor Bean

How arsenic (As) is transported in phloem remains unknown. To help answer this question, we quantified the chemical species of As in phloem and xylem exudates of castor bean (Ricinus communis) exposed to arsenate [As(V)], arsenite [As(III)], monomethylarsonic acid [MMA(V)], or dimethylarsinic acid. In the As(V)- and As(III)-exposed plants, As(V) was the main species in xylem exudate (55%–83%) whereas As(III) predominated in phloem exudate (70%–94%). The ratio of As concentrations in phloem to xylem exudate varied from 0.7 to 3.9. Analyses of phloem exudate using high-resolution inductively coupled plasma-mass spectrometry and accurate mass electrospray mass spectrometry coupled to high-performance liquid chromatography identified high concentrations of reduced and oxidized glutathione and some oxidized phytochelatin, but no As(III)-thiol complexes. It is thought that As(III)-thiol complexes would not be stable in the alkaline conditions of phloem sap. Small concentrations of oxidized glutathione and oxidized phytochelatin were found in xylem exudate, where there was also no evidence of As(III)-thiol complexes. MMA(V) was partially reduced to MMA(III) in roots, but only MMA(V) was found in xylem and phloem exudate. Despite the smallest uptake among the four As species supplied to plants, dimethylarsinic acid was most efficiently transported in both xylem and phloem, and its phloem concentration was 3.2 times that in xylem. Our results show that free inorganic As, mainly As(III), was transported in the phloem of castor bean exposed to either As(V) or As(III), and that methylated As species were more mobile than inorganic As in the phloem.Arsenic (As) is an environmental and food chain contaminant that has attracted much attention in recent years. Soil contamination with As may lead to phytotoxicity and reduced crop yield (Panaullah et al., 2009). Food crops are also an important source of inorganic As, a class-one carcinogen, in human dietary intake, and there is a need to decrease the exposure to this toxin (European Food Safety Authority, 2009). Paddy rice (Oryza sativa) is particularly efficient in As accumulation, which poses a potential risk to the population based on a rice diet (Meharg et al., 2009; Zhao et al., 2010a). Other terrestrial food crops generally do not accumulate as much As as paddy rice; however, where soils are contaminated, relatively high concentrations of As in wheat (Triticum aestivum) grain have been reported (Williams et al., 2007; Zhao et al., 2010b). On the other hand, some fern species in the Pteridaceae family are able to tolerate and hyperaccumulate As in the aboveground part to >1,000 mg kg⁻¹ dry weight (e.g. Ma et al., 2001; Zhao et al., 2002); these plants offer the possibility for remediation of As-contaminated soil or water (Salido et al., 2003; Huang et al., 2004). A better understanding of As uptake and long-distance transport, metabolism, and detoxification is needed for developing strategies for mitigating As contamination, through either decreased As accumulation in food crops or enhanced As accumulation for phytoremediation.The pathways of As uptake by plant roots differ between different As species; arsenate [As(V)] enters plant cells via phosphate transporters, whereas arsenite [As(III)] is taken up via some aquaporins (for review, see Zhao et al., 2009). In rice, a silicic acid efflux protein also mediates As(III) efflux toward stele for xylem loading (Ma et al., 2008). Methylated As species, such as monomethylarsonic acid [MMA(V)] and dimethylarsinic acid [DMA(V)], which may be present in the environment as products of microbial or algal methylation of inorganic As or from past uses of methylated As pesticides, are taken up by rice roots partly through the aquaporin NIP2;1 (for nodulin 26-like intrinsic protein; also named Lsi1; Li et al., 2009). Once inside plant cells, As(V) is reduced to As(III), possibly catalyzed by As(V) reductase(s) such as the plant homologs of the yeast (Saccharomyces cerevisiae) ACR2 (Bleeker et al., 2006; Dhankher et al., 2006; Ellis et al., 2006; Duan et al., 2007). As(III) has a high affinity to thiol (-SH) groups and is detoxified by complexation with thiol-rich phytochelatins (PCs; Pickering et al., 2000; Schmöger et al., 2000; Raab et al., 2005; Bluemlein et al., 2009; Liu et al., 2010). As(III)-PC complexation in roots was found to result in reduced mobility for efflux and for long-distance transport, possibly because the complexes are stored in the vacuoles (Liu et al., 2010). Excess As(III) causes cellular toxicity by binding to the vicinal thiol groups of enzymes, such as the plastidial lipoamide dehydrogenase, which has been shown to be a sensitive target of As toxicity (Chen et al., 2010). The As hyperaccumulating Pteris species differ from nonhyperaccumulating plants because of enhanced As(V) uptake (Wang et al., 2002; Poynton et al., 2004), little As(III)-thiol complexation (Zhao et al., 2003; Raab et al., 2004), and efficient xylem loading of As(III) (Su et al., 2008). Recently, an As(III) efflux transporter, PvACR3, has been found to play an important role in As(III) detoxification by transporting As(III) into vacuoles in Pteris vittata (Indriolo et al., 2010).With the exception of As hyperaccumulators, most plant species have a limited root-to-shoot translocation of As (Zhao et al., 2009). The chemical species of As in xylem exudate have been determined in a number of plant species. As(III) was found to be the predominant species (80%–100%) in the xylem sap of rice, tomato (Solanum lycopersicum), cucumber (Cucumis sativus), and P. vittata even when these plants were fed As(V) (Mihucz et al., 2005; Xu et al., 2007; Ma et al., 2008; Su et al., 2010), suggesting that As(V) is reduced in roots before being loaded into the xylem. In other plant species, such as Brassica juncea (Pickering et al., 2000), wheat, and barley (Hordeum vulgare; Su et al., 2010), As(V) accounted for larger proportions (40%–50%) of the total As in the xylem sap. Studies using HPLC-inductively coupled plasma (ICP)-mass spectrometry (MS) coupled with electrospray (ES)-MS showed no evidence of As(III)-thiol complexation in the xylem sap of sunflower (Helianthus annuus; Raab et al., 2005). When rice plants were exposed to MMA(V) or DMA(V), both As species were found in the xylem sap (Li et al., 2009). Generally, methylated As species are taken up by roots at slower rates than inorganic As, but they are more mobile during the xylem transport from roots to shoots (Marin et al., 1992; Raab et al., 2007; Li et al., 2009).It has been shown that phloem transport contributes substantially to As accumulation in rice grain (Carey et al., 2010). However, little is known about how As is transported in phloem (Zhao et al., 2009). There are no reports on the chemical species of As in phloem exudate. The speciation of As in phloem is important because it dictates how As is loaded in the source tissues and unloaded in the sink tissues, such as grain. Questions with regard to the oxidation state, methylation, and complexation of As in phloem sap remain to be answered. Unlike xylem sap, phloem sap is much more difficult to obtain in sufficient quantities for analysis. In this study, we investigated As speciation in phloem and xylem exudates of castor bean (Ricinus communis), which is widely used as a model plant to investigate phloem transport of solutes (e.g. Hall et al., 1971; Hall and Baker, 1972; Allen and Smith, 1986; Bromilow et al., 1987).     

Tissue-Specific Isoforms of Catalase Subunits in Castor Bean Seedlings

Catalases purified from endosperm glyoxysomes and non-specializedmicrobodies from hypocotyls of castor bean seedlings differedin their specific activity [90–164 and 0.89–4.9kunits (mg protein)^–1, respectively] and in their constituentsubunits [two subunits of 54 and 56 kDa for the endosperm enzymeand only one of 56 kDa for the hypocotyl enzyme]. Immunoblotanalysis also showed that particulate fractions from the endospermsand from etiolated and green cotyledons contained two catalasesubunits of 54 and 56 kDa, whereas such fractions from the hypocotylsand roots contained only the 56-kDa subunit. Leaf peroxisomesfrom green leaves had two catalase subunits of around 55 kDaeach. Results of translation in vitro indicated that the 54-and 56-kDa subunits were translated from distinct mRNAs andlevels of both mRNAs increased in the endosperms during germination,prior to increases in levels of catalase proteins. In the hypocotyls,the 56-kDa subunit seemed to be synthesized constitutively. ¹Present addresses: YO, Toyota Central Institute, 31-9 Musashizuka,Nagabuchi, Nagakute, Aichi 480-11, Japan     

Sucrose-Induced Increase of Invertase Synthesis in Castor Bean Cotyledons

Abstract

Aumento della sintesi di invertasi in seguito a trattamento con saccarosio in cotiledoni isolati da semi germinanti di ricino. – L'attività invertasica per cotiledone aumenta durante la germinazione di semi di ricino. In cotiledoni isolati ed incubati in acqua distillata per 15–22 ore, l'aumento di attività invertasica è molto scarso, L'aggiunta di saccarosio 0,1 M al mezzo di incubazione provoca un aumento di circa 40% dell'attività invertasica; aumento che non si riscontra se i cotiledoni vengono incubati in glucosio 0,1 M. La pre-senza di attinomicina D e di puromicina nel mezzo di incubazione previene lo sviluppo dell'attività invertasica. L'apparente specificità del saccarosio nell'indurre l'aumento di sintesi dell'enzima viene brevemente discussa nel quadro piú ampio dei fenomeni di regolazione da substrato delle sintesi di enzimi.